SUMMARYNoonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome.

Convergence and extension (C&E) cell movements are essential to shape the body axis during vertebrate gastrulation. We have used the zebrafish to assess the role of the receptor protein-tyrosine phosphatases, RPTPalpha and PTPepsilon, in gastrulation cell movements. Both RPTPalpha and PTPepsilon knockdown and ptpra(-/-) embryos show defects in C&E movements. A method was developed to track gastrulation cell movements using confocal microscopy in a quantitative manner and ptpra(-/-) embryos displayed reduced convergence as well as extension speeds. RPTPalpha and PTPepsilon knockdowns cooperated with knockdown of a well known factor in C&E cell movement, non-canonical Wnt11. RPTPalpha and PTPepsilon dephosphorylate and activate Src family kinases in various cell types in vitro and in vivo. We found that Src family kinase phosphorylation was enhanced in ptpra(-/-) embryos, consistent with reduced Src family kinase activity. Importantly, both ptpra(-/-) and RPTPalpha and PTPepsilon knockdown induced C&E defects were rescued by active Fyn and Yes. Moreover, active RhoA rescued the RPTPalpha and PTPepsilon knockdown and ptpra(-/-) induced gastrulation cell movement defects as well. Our results demonstrate that RPTPalpha and PTPepsilon are essential for C&E movements in a signaling pathway parallel to non-canonical Wnts and upstream of Fyn, Yes and RhoA.

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11Cul). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11Cul fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11Cul mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.